Method and device for separating particles or molecules by migration through a ferrofluid

ABSTRACT

The invention concerns a method for separating particles or molecules whereby these particles or molecules are introduced in a separating medium and a moving force is applied to them in said medium. The method is characterised in that the separating medium is a ferrofluid, i.e. a colloidal suspension of magnetic particles and a magnetic field is applied to this ferrofluid generating therein at least an alternation of a zone rich in magnetic particles and a zone poor in magnetic particles, part at least of the region of the ferrofluid in which this alternation is generated is passed through by the particles or molecules to be separated during their migration.

The present invention relates to methods for separating particles ormolecules by migration in a separation medium.

A large number of methods are already known in which separation isobtained by applying a motive force within a separation medium toparticles or molecules to be separated.

If the motive force is of the electrical type, the situation is referredto as electrophoresis, and if it is of hydrodynamic origin, thesituation is referred to as chromatography or filtration.

CONTEXT AND PRIOR ART

Chromatography, filtration and electrophoresis are very widely used forpurifying or analyzing molecules or macromolecules, whether synthetic ornatural, particles, cells, organelles or viruses. There are large numberof ways of implementing these methods, descriptions of which may befound in various works, for example “Chromatographie en phase liquide etsupercritique”, R. Rosset, M. Caude, A. Jardy, Masson ed., Paris 1991;“Practical High Performance Liquid Chromatography, V. R. Meyer, JohnWiley ed. Chichester, N.Y. USA; “Chromatography of polymers”, T. Provdered., ACS publ. Washington D.C., 1993 or “Electrophoresis: theory,techniques and biochemical and clinical applications, A. T. Andrews,Oxford University Press, N.Y. 1986”.

However, these techniques have limitations. For DNA, for example,chromatography only allows separation for molecules of relatively smallsize, and agarose or acrylamide gel electrophoresis is most oftenpreferred to it.

Constant-field electrophoresis does not itself make it possible toseparate molecules larger than a few tens of thousands of base pairs(kilobases, Kb), while the intact chromosomes of most cellularorganisms, prokaryotic or eukaryotic, and much viral DNA, are severalhundreds of kilobases or several megabases (millions of base pairs, Mb)long. Human DNA, for example, has sizes between 50 and 200 Mb. For alarge number of applications in medicine and genetics, such as genetic(genome) mapping, variability analysis, cloning with artificial yeastchromosomes, diagnosis, etc., DNA exceeding the separation limits ofconstant-field electrophoresis needs to be separated as a function ofits size.

In order to solve this problem, a new technique known as pulsed fieldgel electrophoresis has been proposed (PCT WO 84/02001, inventors C.Cantor and D. C. Schwartz, 24.05.84). A large number of variants of thistechnique has also been developed (see for example EP 0 356 187, EP 0256 737, U.S. Pat. No. 4,971,671, EP 0 395 315, “Pulsed filed [sic]Electrophoresis”, B. Birren, E. Lai, Academic Press, London 1993;“Pulsed filed [sic] Gel electrophoresis”, Meth. in Mol. Biol., M.Burmeister and L. Ulanovsky Eds, Humana Press, Totowa, N.J. USA, 1992,and the references cited in these patents and works).

In spite of its significant success, this technique, too, still hasdrawbacks. The main one is its slowness: it takes several days toseparate chromosomes containing several megabases. In addition, sincethe separation is carried out in a gel, it is difficult to recover theDNA after separation, and this method is poorly suited to preparativeapplications. Lastly, it remains limited to sizes smaller than 10 Mb.

Electrophoresis can also be used to separate particles of micron orsubmicron size (colloidal particles, cells, viruses, red blood cells orwhite blood cells, etc.), as may be necessary in sample analysis, inpurification, for diagnosis or for treating certain diseases.

If the particles to be separated have different surface potentials, theycan be separated in a liquid medium as a function of this surfacepotential. Conversely, for a large number of applications it isdesirable to separate them as a function of the size of the particles ormolecules having the same surface potential. This cannot be done in aliquid medium, and it has been proposed to carry out this type ofseparation by electrophoresis in very dilute agarose gel, but theparticles have a tendency to become trapped in the gel, and these gelsare very difficult to handle. Further, this method only works forrelatively small particles, typically smaller than one micrometer (G. A.Griess, P. Serwer, Biopolymers, 29, 1863-1866 (1990)). In this case aswell, pulsed field electrophoresis makes it possible to extend the rangeaccessible to the method slightly, but in a limited way. Lastly, itshould be pointed out that it does not make it possible for particles ofsimilar size to be separated satisfactorily.

In summary, although methods are already known for separating particles,large molecules and in particular DNA, these methods have a large numberof drawbacks, linked in particular with their slowness and thedifficulties which are encountered with these methods for carrying outthe separation of molecules or particles of large size.

One object of the invention is to provide a separation method which doesnot have these various drawbacks.

Separation methods are already known which employ a combination ofelectric and magnetic fields in order to separate particles, and inparticular DNA.

These methods, referred to as electromagnetophoresis, have beendescribed for example in U.S. Pat. No. 4,726,904 and also in thefollowing publications:

Mukherjee, H. G., majumdar, D., “Fresenius'Z” Anal. Chem., 277, 205(1975),

O. Lumpkin, J. Chem. Phys. 92, 3848-3852 (1990)

Kowalczuk, J. S., Acta Chromatogr. 1, 34-55 (1992).

In these methods, the magnetic field supplements the electric field tocause migration of the charges to be separated.

U.S. Pat. No. 4,526,681 has already proposed a technique for separatingmagnetic particles, according to which the particles to be separated areintroduced into a ferrofluid medium, to which a magnetic fields [sic] isapplied which makes it possible to distribute said particles along amagnetic susceptibility gradient.

However, this technique can only be applied for the separation ofmagnetic particles having different magnetic susceptibilities.

OBJECTS OF THE INVENTION

For its part, the invention provides a method for separating particlesor molecules, in which these particles or molecules are introduced intoa separation medium which is a ferrofluid, that is say a colloidalsuspension of magnetic particles, and at least one motive force isapplied to these particles or molecules within said ferrofluid,characterized in that a magnetic field is applied to this ferrofluidwhich creates in it at least one alternation of one (or more) zone(s)rich in and a zone lean in magnetic particles that the particles ormolecules pass through during their migration, which brings about theirseparation.

A method of this type makes it possible to separate the particles ormolecules as a function of the speed at which they move within theferrofluid, and performs better than the methods known to date in termsof separation speed and/or in terms of the size range in which theseparation can be carried out.

It will be noted that the method proposed by the invention makes itpossible to separate essentially nonmagnetic particles (minimal or zeromagnetic susceptibility).

The motive or migration force used is generally nonmagnetic.

In a preferred embodiment of the invention, which is particularlyadvantageous for the separation of particles carrying an electriccharge, the migration force is obtained by applying an electric fieldwithin the separation medium. The method then constitutes anelectrophoresis method.

It will in general be advantageous to choose as the separation medium aferrofluid whose magnetic particles are essentially neutral, so thatthey will not be moved under the action of the electric field. However,certain particular applications may nevertheless require magneticparticles of given charge, if it is desired for example to decrease orincrease the interaction of the particles to be separated with theparticles.

In a preferred embodiment, which may or may not be combined with thepreferred embodiments described above, the magnetic field is essentiallyperpendicular to the direction of motion of the particles.

Two embodiment versions which are moreover preferred, and may becombined with any one of the embodiments described above but aremutually exclusive, consist in using a magnetic field:

a) which is essentially constant in the zone where it is applied,

b) which has an intensity gradient in the zone where it is applied.

Similarly, two embodiment versions which may be combined with any one ofthe embodiments described above but are mutually exclusive, consist inusing:

c) a separation zone which has a thickness which is essentially constantin the direction of the magnetic field, this thickness being chosen,with the concentration of the magnetic particles in the separation fluidand the amplitude of the magnetic field, as a function of the dimensionsof the particles or molecules to be separated; in particular, largerthicknesses are preferably used for particles and molecules of largersize;

d) the separation zone has a variable thickness along the preferentialmigration direction of the particles or molecules to be separated, thethickness of this zone being chosen, with the concentration of themagnetic particles in the separation fluid and the amplitude of themagnetic field, as a function of the dimensions of the particles andmolecules to be separated; in particular, larger thicknesses arepreferably used for particles and molecules of larger size.

The methods according to paragraphs a) and c) are preferably used toobtain a high resolution over a relatively small range of sizes, whilethe methods according to paragraphs b) and d) are better suited toseparations in a large range of sizes.

In a particularly simple embodiment variant of paragraph d), the wallsof the separation zone which are essentially perpendicular to themagnetic field are slightly inclined relative to one another, givingsaid zone a “wedge” shape.

In the favored embodiments described above, the best results are mostoften obtained when the average dimension of the separation zone in thedirection parallel to the direction of the magnetic field is between 1micrometer and 1 mm, and preferably between 10 micrometers, and 100micrometers.

This method is advantageously implemented with the various followingsteps:

filling the separation zone with the separation medium;

activating the magnetic field;

introducing a certain quantity of a sample containing the particles ormolecules to be separated on one side of the separation zone;

activating means exerting a motive force on the particles or moleculesto be separated.

It should be noted that the order in which these steps are listedcorresponds to a favored embodiment of the invention, but that it isequally possible in the scope of the invention to implement them in adifferent order, for example by activating the magnetic field after theintroduction of the samples to be separated, and/or by activating ratherthe motive force.

In the scope of the invention, detection or observation of the passingof the separated products and/or collection of the separated productsmay be implemented at the outlet of the separation zone.

Advantageously, in addition, the ferrofluid is automatically replacedbetween two separation operations.

The invention is particularly advantageous for the separation ofparticles or macromolecules of large size, such as nucleic acids andparticularly DNA, and yet more particularly DNA molecules of sizebetween 50 Kb and several hundreds of Mb. It is also particularlyadvantageous for the separation of objects in suspension in a liquid,such as cells, viruses, nonmagnetic colloidal suspensions and liposomes.These examples should not however be interpreted as a restriction of thefield of the invention, which may also in certain cases proveadvantageous for the separation of other types of objects like, hereagain given by way of nonrestrictive examples, proteins and synthetic ornatural macromolecules.

A further object of the invention is to provide a device for theseparation of particles or molecules which comprises a cell thatreceives a separation medium, which is a ferrofluid, that is to say acolloidal suspension of magnetic particles, means for introducing theseparticles or molecules into said separation medium and means forapplying a magnetic field to this fluid, characterized in that itincludes means for applying at least one migration force within saidmedium and in that, for the application of a magnetic field, there aremeans capable of creating at least one alternation of a zone rich in anda zone lean in ferrofluid magnetic particles that the particles ormolecules to be separated pass through during their migration, whichbrings about their separation.

The method and the device according to the invention are advantageouslyused in the scope of a diagnostic method, for separating particles andmolecules and, especially but not exclusively, DNA, cells, blood cellsor viruses.

They may also be used for obtaining medicines, veterinary orphytosanitary products and cosmetic products, for example includingliposomes, proteins, DNA, cells, blood cells, viruses or colloidalsuspensions in their composition.

Other characteristics and advantages of the invention will become moreapparent from the following description. This description is purelyillustrative and does not imply any limitation.

PRESENTATION OF THE FIGURES

This description should be read with reference to the appended drawings,in which:

FIG. 1 is a schematic representation in plan view of an example of adevice for implementing the invention;

FIG. 2 is a schematic sectional view of the device in FIG. 1;

FIG. 3 represents a preferred embodiment of a variant of a deviceaccording to the invention similar to the one in FIGS. 1 and 2;

FIGS. 4a to 4 d are microphotographs illustrating examples of migration;

FIGS. 5a and 5 b are graphs on which two examples of the variation inthe fluorescence at the outlet of a separation device according to theone in FIG. 3 as a function of time have been plotted.

FIG. 6 is a schematic sectional view of the device in FIG. 1.

DETAILED DESCRIPTION

FIGS. 1 and 2 illustrate a possible device for implementing theinvention.

This device comprises an essentially parallelpipedal channel 1 whichjoins two reservoirs 2 and 3 and into which a ferrofluid, that is to saya liquid containing a colloidal suspension of magnetic particles, isintroduced before the separation phase.

This channel 1 constitutes the separation zone.

The device includes means for creating a magnetic field, essentiallyparallel to the thickness e of said channel, in at least one part ofsaid channel 1.

The effect of this magnetic field is to organize the ferrofluid, inwhich columns 26 rich in magnetic particles and one or more zones leanin magnetic particles become created. Various means capable of creatingsuch a magnetic field are known to the person skilled in the art, forexample Helmoltz coils, electromagnets or permanent magnets.

For most applications, it is advantageous to apply an essentiallyuniform magnetic field in the separation zone. One simple way ofproducing such a field, which is schematically represented in FIG. 2, isto place the poles 7 and 8 (respectively North and South) of a permanentmagnet or of an electromagnet on either side of the separation channel1. Another way, schematically represented in FIG. 3, consists inconstructing a channel with an axis that is essentially circular andconcentric with a Helmoltz coil 37 powered by a current generator (notshown). (All the other elements of the device have functions similar tothose described with reference to FIG. 1).

It should, however, be noted that certain applications may require anonuniform field. The reason for this is that, as is known (E. M.Lawrence et al., Int. J. of Modern Phys. B, 8, 2765-2777, 1994) the sizeand the spacing of the columns rich in magnetic particles depend on themagnetic field and the thickness of the cell. It is therefore possibleto act right away [sic] magnetic as a function of the desiredseparation.

In a large number of cases, in particular when the particles to beseparated are charged, for example if they are polyelectrolytes such asDNA or proteins, cells or viruses, it is advantageous to drive them bymeans of an electric field essentially perpendicular to the magneticfield used for structuring the separation medium.

To that end, the device includes two electrodes 4 and 5 which areconnected to the terminals of a voltage or current generator 6 and arerespectively immersed in the reservoirs 2 and 3. Said generator 6 canoperate in constant current mode, in constant voltage mode, in constantdissipated power mode, or deliver a current or a voltage having a morecomplicated profile.

In particular, it may be advantageous in certain cases, and inparticular for separating DNA molecules of very large size, to use avoltage whose sense or direction changes repetitively with time,according to the principle of pulsed field electrophoresis, which iswell known to the person skilled in the art and is described for examplein “Pulsed field Electrophoresis”, B. Birren, E. Lai, Academic Press,London, 1993; “Pulsed filed [sic] gel electrophoresis”, Meth. In Mol.Biol., M. Burmeister and L. Ulanovsky, Humana Press, Totowa, N.J. USA1992.

Also in general, the electrode with the same polarity as the particlesto be separated is placed on the side of the separation zone 1 via whichthe sample is introduced, but this arrangement may sometimes be reversedif strong electro-osmosis takes place in the cell, for example if themagnetic particles of the ferrofluid are charged and of the same sign asthe particles to be separated.

In order to remove the gases which maybe formed at the electrodes 4 and5, the reservoirs 2 and 3 are advantageously connected to the freeatmosphere or to a common reservoir, either directly or via vents 16 and17.

The device used for implementing the invention may further optionallyinclude a sample introduction zone 9. One configuration which isadvantageous for simultaneously producing the separation zone and thesample introduction zone, consists in obtaining the volume in which theseparation takes place by a process of etching or molding on aninsulating substrate (for example glass or a plastic) and in using, inorder to introduce a well-defined quantity of sample, an auxiliarychannel 23 which is etched on the same substrate and is joined to one ormore reservoirs or ducts 24, 25, between which it is possible to imposea pressure difference or an electric potential difference, for exampleusing two optional electrodes 27 and 28 powered by a generator 29, asdescribed, for example in A. T. Woolley et al., Proc. Natl. Acad. SciUS, 91, 11348-11352 (1994).

The means for introducing the sample may also, for example, consist:

of one or more indentations or “wells” in which the sample is deposited,as in gel electrophoresis “Electrophoresis: theory, techniques andbiochemical and clinical applications, A. T. Andrews, Oxford UniversityPress, N.Y. 1986”,

or alternatively of a pressurizing or depressurizing system as incapillary electrophoresis, see for example “Capillary Electrophoresis”,P. D. Grossman, J. C. Colburn eds, Academic Press, San Diego, Calif.,USA, 1992),

or alternatively of a channel through which a trickle of sample ispoured continuously, as in liquid stream electrophoresis,

or alternatively of one of the sample introduction methods employed inchromatography (“Chromatographie en phase liquide et supercritique”, R.Rosset, M. Caude, A. Jardy, Masson ed., Paris 1991; “Practical HighPerformance Liquid Chromatography, V. R. Meyer, John Wiley ed.Chichester, N.Y. USA; “Chromatography of polymers”, T. Prodver ed., ACSpubl. Washington D.C., 1993).

This list is not, of course, exhaustive.

Similarly, a large number of means capable of detecting the separatedproducts may optionally be combined with the invention, on the side ofthe channel 1 opposite its inlet (outlet window 10). A large number ofdetectors employed in chromatography, in electrophoresis or otherwiseare known to the person skilled in the art (“Capillary Electrophoresis”,P. D. Grossman, J. C. Colurn eds, Academic Press, San Diego, Calif.,USA, 1992) and may be used in the scope of the invention: detection byabsorption of visible or ultraviolet light, by fluorescent orluminescent emission or irradiation emitted by a radioactive substance,by conductimetry or alternatively by scattering of light, etc.

The embodiment presented in FIG. 2 corresponds to detection byfluorescence; the device includes a source 13 which emits light that canbe absorbed by the particles to be detected 20 and is focused by a lens11 in the detection window 10. Said particles re-emit fluorescent light14 which has a longer wavelength than the incident light 12 and is splitfrom it by a dichroic filter 15, then is detected by a photosensitivedetector 21 optionally connected to an analysis system 22.

The cell may also advantageously, although not necessarily, be equipped,preferably close to its outlet face, with one or more devices intendedto collect the various separated products. Such fraction collectionsystems are known to the person skilled in the art and are already usedin chromatography and in electrophoresis. They have not been representedin the figure for the sake of clarity. They may be, for example, aseries of tubes collecting the products at various points in the cell,in order to distribute them into various containers (K. Hannig,Electrophoresis, 3, 235-243 (1982)), or conversely a single tube whichpours the products leaving the cell at different times into two variouscontainers (see for example R. Grimm, J. Cap. Elec. 2, 111-115 (1995),or alternatively a membrane which moves past close to the outlet of thecell and is capable of adsorbing one or more of the separated products(K. O. Eriksson, A. Palm, S. Hjerten, Anal. Biochem., 201 211-215(1992)).

The mode of operation of the invention according to the preferredembodiment presented in FIG. 3 will now be described.

First, the separation zone 1 is filled with a ferrofluid, using forexample capillary action or suppression [sic] in one of the reservoirs 2or 3. The next step involves progressively applying, using the Helmoltzcoil 37, a magnetic field sufficient to order the ferrofluid, accordingto the mechanism described in the aforementioned publication by Lawrenceet al. (1994): under the field action, each magnetic particle of theferrofluid is converted into a micromagnet, and these magnets becomegrouped together to form regularly spaced columns rich in magneticparticles and parallel to the magnetic field.

The sample containing the particles to be separated, for example DNAmolecules, is then introduced. If the sample is of the solid type, suchas for example an agarose insert well known to the person skilled in theart, it may simply be placed in the reservoir 25 using a microspatula.If it is liquid, it may be introduced into said reservoir using amicropipette, or using a tube. The next step is to apply slightpressurization between the reservoirs 25 and 24, or an electricpotential difference between said reservoirs, using the optionalelectrodes 27 and 28. Said pressurization or said electric potentialdifference have the effect of inducing migration of the molecules of theparticles contained in the sample from the reservoir 25 to the reservoir24 via the channel 23, and in particular of leading to the formation ofa well-defined sample zone 9 at the inlet of the separation zone 1.

The introduction is then stopped, and the device creating a migrationforce on the particles to be separated (generator 6, electrodes 7 and 8)is activated. Under the action of this force, the particles to beseparated (20) penetrate the separation zone 1. During their migrationin this zone, they are separated and the various products initiallypresent in the sample can be identified by their migration time usingthe detector 21, or can be collected.

An explanatory model of the separation technique which the inventionprovides will be put forward below. This model is given to assistunderstanding of the invention and is in no way intended to beexhaustive and restrictive.

During their migration, the particles to be separated which arecontained in the sample encounter the columns of magnetic particles, thecohesion of which is ensured by the presence of the field. The particlesneed to get round these obstacles in order to be transported, and aretherefore slowed. It will be understood that this slowing depends on thesize of the particles, the largest particles being braked the most.

Various possible mechanisms for such slowing have already been proposedin the context of gel electrophoresis (see for example G. W. Slater etal. Biopolymers 27, 509-524 (1988)) or in that of electrophoresis inmicrolithographic networks (see for example E. M. Sevick and D. R. M.Williams, Phys. Rev. Lett., 76, 2595-2598 (1996)). However, theinvention presents several advantages over these methods of the priorart:

a/ The maximum size of the particles which can be separated is linkedwith the size of the pores. Within a gel, this pore size is difficult tocontrol, and in particular it is difficult or even impossible to preparegels having a pore size larger than a few tenths of a micrometer. In thecase of microlithography, it is difficult to construct thick cells ofmore than about 10 microns, which limits sensitivity, flow rate and easeof use. In the scope of the invention, however, by varying the thicknessof the cell, the magnetic particle concentration of the ferrofluid andthe amplitude of the magnetic field, it is possible to vary at will thedistance between the columns, which defines the pore size, and inparticular to separate particles of larger size than with a gel. Thus,larger pores and therefore a thicker cell will be chosen in order toseparate particles of larger size.

It will therefore be understood that, in order to obtain optimumresolution in a moderate size range, it is beneficial to have aseparation zone of constant thickness and a magnetic field of uniformintensity. However, it may also be advantageous for certainapplications, in particular when the separation of particles in a largesize range is involved, to use a cell of variable thickness, obtainedfor example by giving a “wedge” shape to the zone in which theseparation is carried out.

b/ Since the distance between the obstacles to the progress of theparticles to be separated can be rendered at will larger in the scope ofthe invention than in gel electrophoresis, friction is reduced and theseparation speed can be much higher.

c/ In the case of the invention, as in that of microlithography, theobstacles form a well-ordered network with a very uniform repeat unit,which leads to lower velocity dispersion and therefore better resolutionthan in a gel which has a less regular and uncontrollable structure. Incontrast to microlithography, in the scope of the invention it is alsopossible to vary the size and/or spacing of the obstacles withoutchanging the separation cell itself.

d/ In the scope of the invention, the network of obstacles responsiblefor the separation can be destroyed and reformed at will, for example inorder to use pressurization to remove a contaminated separation mediumfrom the separation cell and replace it with a fresh medium, or in orderto avoid the trapping of particles within said medium. Inelectrophoresis, however, the obstacle network can only be removed fromthe device manually once it has been formed. Lastly, in lithographicnetworks, the network of obstacles is permanent, and it can only bereplaced by replacing the cell, which considerably increases the runningcost, since the manufacturing cost of these cells is very high.

e/ If desired, certain properties of the separation medium such as therigidity or the dimension of the obstacles may be varied at will duringthe separation by modifying the magnetic field, while these propertiesare immutable and uncontrollable in the case of gel or microlithographicnetwork electrophoresis.

f/ Lastly, it may be pointed out that the magnetic particles can beremoved very easily from the solution after separation in order torecover a purified product, for example using a magnet, while in thecase of a gel, eliminating the agarose requires tricky and moreexpensive digestion by agarase.

EMBODIMENT EXAMPLE

Preparation of the Ferrofluid Emulsion

The emulsion is prepared using the procedure published by J. Bibette inJ. Magn and Magn. Mat. v. 122, p 37 (1993) and J. Coll. and Int. Sci v.147, p 474 (1991). In short, the emulsion is obtained under shear in agrinder from a 50% (w/w) water-SDS solution, into which a solution offerrofluids from Rhône Poulenc containing 20 nm FE₂O₃ [sic] grains in anoil/Fe₂O₃ ratio of 50% (w/w) is progressively incorporated, untilreaching a final oil/water ratio of 80% (w/w). This solution is dilutedten times in water. The ferrofluid drops are then sedimented under amagnetic field, the supernatant is taken off and the ferrofluid emulsionis re-suspended in a 0.05% (w/w) solution of Tergitol type NP10(Sigma)/water. This rinsing operation is repeated four times. The resultis a ferrofluid emulsion having an oil/water interface with negligiblesurface electric charge. Before electrophoresis, this emulsion issupplemented by a TBE buffer solution (45 mM TRIS, 45 mM boric acid,1.25 mM EDTA, pH 8.3, concentrated 20 times. The final ferrofluidemulsion solution therefore has an oil/water ratio of 8% (w/w) andcontains 0.05% (w/w) of NP10, TBE (45 mM TRIS, 45 mM boric acid, 1.25 mMEDTA) at pH 8.3.

Production of the Electrophoresis Cell

The channel 1 is an annular capillary of thickness chosen between 0.01and 0.05 mm, 4 mm in width and 24 mm in length. It is produced,according to the general scheme presented in FIG. 3, by depositing afilm of “parafilm” (American National Cup) stretched manually over around glass plate of diameter 32 mm and having four perforations passingentirely through it, which respectively fulfil the role of reservoirs 2,3, 24 and 25. The separation channel 1 and the sample introductionchannel 23 are formed by etching on the “parafilm” and are closed byapplying a round microscope slide of diameter 30 mm on said film. Thecell is sealed by applying previously heated paraffin on its periphery.The cell thus formed is then placed in the magnetic device 37, so thatthe detection system 11 lies facing a zone 10 of the main channel 1close to the outlet 3 of the latter, with the filling orifices 2, 3, 24and 25 on top, and the electrodes 4, 5 (and optionally 27 and 28) areplaced in said orifices.

The cell is filled by capillary action with the magnetic emulsiondescribed above, placed in the reservoir 3. A 5 mT magnetic field isprogressively applied (200 mT/minute) to the electrophoresis cell placedinside the electromagnet. The axis of the electromagnet coincides withthat of the 30 mm circular slide of the electrophoresis cell. Thisprocedure ensures the formation of a regular array of ferrofluid columnswhose average spacing, which is a function of the thickness of the cell,is from 0.002 to 0.01 mm (see Lawrence et al. International Journal ofModern Physics B, v. 8, p 2765 (1994). The diameter of the columns canbe modulated by the oil concentration of the ferrofluid solution. Atransverse channel 23, joining two reservoirs 24 and 25 is used forcontrolled introduction of the sample. Once the network of columns offerrofluids have been formed [sic] by setting up the magnetic field, apiece of gel, or a liquid aliquot containing the chromosomes to beseparated, (incubated beforehand for at least four hours in a 0.005 mMsolution of the YOYO fluorescent intercalating agent (Molecular Probes)if it is desired to carry out detection by fluorescence), is placed inthe reservoir 25 and the pressures are allowed to equilibrate in thecell. The transverse channel 23 is then filled with a solutioncontaining the DNA to be separated, by setting up an electric potentialdifference between the electrodes 26 and 27, or possibly a hydrostaticpressure difference, between the reservoirs 25 and 24; once the channel23 has been filled, the potential difference between the reservoirs 24and 25 is terminated and a potential difference (typically a few tens ofvolts) is applied between the reservoirs 2 and 3, by means of theelectrodes 4 and 5 (in the case of separating negative species such asDNA, and in the presence of essentially neutral ferrofluids, theelectrode 4 is at the negative potential). This method makes it possibleto introduce a well-defined sample volume, corresponding approximatelyto the center of the cross (volume 9).

Observation of the DNA Molecules During the Electrophoresis

This observation is not necessary for the separation, but it is usefulfor understanding the mechanisms responsible for said separation. It iscarried out with a Nikon Diaphot-TMD-EF epi-fluorescence invertedmicroscope with an immersion objective having a magnification of 100×,equipped with an image intensifier system (Hamamatsu) connected to a CCDcamera and to a display system (Hamamatsu).

The electrophoresis cell described above is mounted on the microscopeusing a suitable circular support placed inside a cylindricalelectromagnet making it possible to obtain 5 mT magnetic fields. The DNAmolecules are observed by the fluorescent emission at wavelengths longerthan 520 nm from the YOYO (Molecular Probes) intercalated in the DNAmolecules and excited by light of wavelength between 450 and 490 nm. Themigration of the DNA molecules inside the ferrofluid structure isrecorded on video cassettes.

FIGS. 4a to 4 d show examples of migration of S. Cerevisae chromosomes.Observing the video sequences clearly shows the following essentialpoints:

the ferrofluid columns are motionless between certain electric andmagnetic field limits (here, for a 5 mT magnetic field a voltage of morethan 10 V can be applied to the terminals of the capillary).

the DNA molecules penetrate and move between the ferrofluid structures.

the DNA molecules scatter round the ferrofluid columns without any signof strong interaction (adsorption) and without any sign of significantperturbation to the ferrofluid structures.

the shortest chromosomes (˜250,000 base pairs (bp)) retain anessentially spherical configuration on, the observation time scale (40ms) (4 a). This suggests the possibility of separation as a function ofmolecular mass according to a molecular sieve mechanism, known by thename of “Ogston sieving”.

the chromosomes of intermediate size are slowed by the obstacles andtemporarily become stretched in the direction of the electric field (4b).

the longest chromosomes observed (4 c- 4 d) are continuously stretchedin the direction of the electric field. They form U-, J- and I-shapedstructures which are well known in conventional gels. The migrationdynamics of these molecules resemble the migration dynamics of DNAmolecules of small size (20,000˜50,000 bp) in conventional agarose gels.In conventional gels, there is still separation when structures of thistype are observed, which suggests that it should be possible, in acontinuous field, to separate chromosomes of a size extending at leastup to 1,000,000 bp. It is, however, also clear that the use of pulsedfield methods should be feasible. Because of the high mobility of themolecules in the ferrofluid structures compared with their mobility inconventional gels, these pulsed field methods in ferrofluid structuresshould be much faster than in agarose gel.

the ferrofluid structures can be made and unmade at will in the presenceof DNA molecules. This allows new separation methods to be envisaged,which are based on coupling between the ferrofluid structure formationdynamics and the DNA deformation dynamics.

it is possible to make the ferrofluid structures mobile, for example byreducing the intensity of the magnetic field or by omitting the steps ofwashing with Tergitol, i.e. by modifying the surface charge of theferrofluid droplets. This makes it possible to envisage separationmethods based on coupling between the relative mobility of theferrofluid structures and that of the DNA.

Obtaining Elution Profiles

The microscopy set-up was used here to establish electropherograms(profiles of DNA concentration as a function of time) by integrationthroughout the image with respect to time. However, other detectiondevices known to the person skilled in the art could be used to do this.

FIG. 5a represents the profile obtained with the injection of a samplecontaining only lambda phase [sic] DNA at 20 V/cm over a migrationdistance of 20 mm. The first peak corresponds to degradation products(which are difficult to quantify in gel electrophoresis) and the secondpeak to the intact chromosomes.

FIG. 5b represents the profile obtained by mixing the sample containinglambda phage DNA (48.5 Kb) and T2 phage DNA (140 Kb). The first twopeaks emerge after a time identical to that observed in 5 a, and thethird peak is that of T2. This separation is obtained in ½ hour, whileseveral hours are necessary with a pulsed field on gel.

Another embodiment is illustrated in FIG. 6, which is a possible devicefor implementing the invention. The device comprises an inclined channel1 which joins two reservoirs 2 and 3 and into which a ferrofluid, thatis to say a liquid containing a colloidal suspension magnetic particles,is introduced before the separation phase. This channel 1 constitutesthe separation zone. The device includes means for creating magneticfield, adjacent to the changing thickness e of the channel, in at leastone part of said channel 1. The effect of this magnetic field is toorganize the ferrofluid, in which columns 26 rich in magnetic particlesin one or more zones lean and magnetic particles become created. Variousmeans capable of creating such a magnetic field are known to the personskilled in the art, for example Helmoltz coils, electromagnets, orpermanent magnets. For most applications, it is advantageous to apply anessentially uniform magnetic field in the separation zone. One simpleway of producing such a field, which is schematically illustrated inFIG. 6, is to place the poles 7 and 8 (respectively north and south) ofa permanent magnet or of an electromagnet on either side of the inclinedseparation channel 1. (All the other elements of the device havefunctions similar to those described with reference to FIGS. 1 and 2.)

What is claimed is:
 1. Method for separating particles or molecules, in which these particles or molecules are introduced into a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles and at least one motive force is applied to these particles or molecules within said ferrofluid, wherein a magnetic field is applied to this ferrofluid which creates in it at least one alternation of one zone rich in and one zone lean in magnetic particles of the ferrofluid that the particles or molecules to be separated pass through during their migration, which brings about their separation, wherein the migration force is obtained by applying an electric field within the separation medium, said method constituting an electrophoresis method.
 2. Method according to claim 1, wherein the magnetic particles of the separation fluid are substantially neutral.
 3. Method according to claim 1 wherein the magnetic field is substantially perpendicular to the direction of motion of the particles or molecules to be separated.
 4. Method according to claim 1 wherein the magnetic field is substantially constant in the zone where it is applied.
 5. Method according to claim 1 wherein the magnetic field has an intensity gradient in the zone where it is applied.
 6. Method according to claim 1, wherein the separation zone has a thickness which is substantially constant in the direction of the magnetic field, this thickness being chosen, as a function of a dimension of the particles or molecules to be separated.
 7. Method according to claim 1, wherein the separation zone has a variable thickness along the preferential migration direction of the particles or molecules to be separated, the average thickness of this zone being chosen, as a function of the dimensions of the particles or molecules to be separated.
 8. Method according to claim 1, wherein at least two opposite walls of the separation zone are substantially inclined relative to one another.
 9. Method according to claim 1 wherein the thickness of the separation zone is between 1 μm and 1 mm.
 10. Method according to claim 9, wherein the thickness of the separation zone is between 10 μm and 100 μm.
 11. Method according to claim 1 wherein the following steps, are carried out in an arbitrary order: filling the separation zone with the separation medium; activating the magnetic field; introducing a certain quantity of a sample containing the particles or molecules to be separated on one side of the separation zone; exerting a motive force on the particles or molecules to be separated.
 12. Method according to claim 11, wherein the filing of the separation zone with the separation medium precedes the activation of the magnetic field.
 13. Method according to claim 11, wherein at the outlet of the separation zone, at least one of the passing of the separated products is detected and the separated products are collected.
 14. Method according to claim 1 wherein the application of the magnetic field precedes the sample introduction.
 15. Method according to claim 1 wherein the sample introduction precedes the step of exerting a motive force on the particles or molecules to be separated.
 16. Method according to claim 1 wherein the colloidal fluid of magnetic particles is automatically replaced between two separation operations.
 17. Method according to claim 1 wherein the molecules separated are nucleic acids.
 18. Method according to claim 17, wherein the molecules separated are DNA molecules whose size is between 50 Kb and several hundreds of Mb.
 19. Method according to claim 1 wherein the particles or molecules separated are at least one of cells, viruses, nonmagnetic colloidal suspensions and liposomes.
 20. Method according to claim 1, wherein the particles or molecules to be separated are essentially non-magnetic.
 21. Method according to claim 1, wherein the creation of said at least one alternation of zone rich in and zone lean in magnetic particles by means of said magnetic fields is created before the particles of the molecules to be separated encounter said magnetic particles.
 22. Method according to claim 1, wherein said at least one alternation of zone rich in and zone lean in magnetic particles is created by assembling in the separation medium a multiplicity of columns rich in magnetic particles which the molecules or particles to be separated encounter during their migration.
 23. Method according to claim 22, wherein the columns are substantially parallel to the magnetic field.
 24. Method according to claim 22, wherein the columns are regularly spaced.
 25. Method according to claim 1, wherein one or several separated particles or molecules are detected.
 26. Method according to claim 1, wherein the particles or molecules to be separated are proteins, natural macromolecules or synthetic macromolecules.
 27. Device for the separation of particles or molecules, in which the particles or molecules are selected from the group consisting of liposomes, proteins, DNA, cells, blood cells, synthetic macromolecules, natural macromolecules, and viruses, comprising a cell including a separation medium with a ferrofluid having a colloidal suspension of magnetic particles, means for introducing these particles or molecules into said separation medium and means for applying a magnetic field to this fluid, said device further comprising means for applying at least one migration force within said medium and means for creating at least one alternation of a zone rich in and a zone lean in ferrofluid magnetic particles that the particles or molecules to be separated pass through during their migration, which brings about their separation, said device further including means to collect at least one separated particles or molecules.
 28. Device for the separation of particles or molecules which comprises a cell that contains a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles, means for introducing these particles or molecules into said separation medium and means for applying a magnetic field to this fluid within said medium, wherein said means for applying the magnetic field comprises means for applying at least one migration force within said medium and means for creating at least one alternation of a zone rich in and a zone lean in ferrofluid magnetic particles that the particles or molecules to be separated pass through during their migration, which brings about their separation, said device further including means to detect the separated particles or molecules when they have passed through said alternation, wherein the migration force is obtained by applying an electric field within the separation medium, the device constituting an electrophoresis device.
 29. Device according to one of claim 28 or 27 wherein the cell is formed by at least one of etching and molding of an insulating substrate.
 30. Device according to claim 28, wherein the thickness of the separation zone is between 1 μm and 1 mm.
 31. Device according to claim 28, wherein the thickness of the separation zone is between 10 μm and 100 μm.
 32. Method for separating particles and molecules, in which the particles or molecules are introduced into a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles and at least one motive force is applied to these particles or molecules within the ferrofluid, wherein a magnetic field is applied to the ferrofluid which creates in it at least one alternation of one zone rich in and one zone lean in magnetic particles of the ferrofluid that the particles or molecules to be separated pass through during their migration, which brings about their separation, wherein at least two opposite walls of the separation zone are substantially perpendicular to the magnetic field and wherein the separation zone has a thickness which is substantially constant in the direction of the magnetic field, the thickness being chosen, as a function of a dimension of the particles or molecules to be separated.
 33. Method for separating particles or molecules in which the particles or molecules are introduced into a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles, and at least one motive force is applied to these particles or molecules within said ferrofluid, wherein a magnetic field is applied to the ferrofluid which creates in it at least one alternation of one zone rich in and one zone lean in magnetic particles of the ferrofluid that the particles or molecules to be separated pass through during their migration, which brings about their separation, wherein the thickness of the separation zone is between 1 μm and 1 mm, and an essentially parallelpipedal channel comprises the separation zone.
 34. Method according to claim 33, wherein the thickness of the separation zone is between 10 μm and 100 μm.
 35. Device for the separation of particles or molecules which comprises a cell that contains a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles, means for introducing these particles or molecules into said separation medium, and means for applying a magnetic field to this fluid within said medium, wherein said means for applying the magnetic field comprise means for applying at least one migration force within said medium and means for creating at least one alternation of a zone rich in and a zone lean in ferrofluid magnetic particles that the particles or molecules to be separated pass through during their migration, which brings about their separation, said device further including means to detect the separated particles or molecules when they have a passed through the alternation, wherein two opposite walls for the separation zone are substantially perpendicular to the magnetic field, and wherein the separation zone has a thickness which is substantially constant in the direction of the magnetic field, the thickness being chosen as a function of a dimension of the particles or molecules to be separated.
 36. Device for the separation of particles or molecules which comprises a cell that contains a separation medium comprising a ferrofluid, having a colloidal suspension of magnetic particles, means for introducing these particles or molecules into said preparation medium and means for applying a magnetic field to this fluid within said medium, wherein said means for applying the magnetic field comprise means for applying at least one migration force within said medium, and means for creating at least one alternation of a zone rich in and a zone lean in ferrofluid magnetic particles that the particles or molecules to be separated pass through during their migration, which brings about their separation, said device further including means to detect the separated particles or molecules when they have passed through said alternation, wherein at least two opposite walls of the separation zone are substantially inclined relative to one another. 